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Embryos were euthanized using a 300 mg/L tricaine methanesulfonate (Merk Life Science S.r.l., Milano, Italy) solution [33 (link)] and fixed in 3.5% formaldehyde/0.1 M sodium phosphate buffer. Soon after, double acid-free staining with Alcian Blue 8GX (Sigma) and alizarin red S (ARS, Sigma) [34 (link)] was applied to stain cartilage and bone tissue, respectively. The quantification of osteogenesis level was measured as vertebral mineralization rate (N.V./L.), calculated as the number of mineralized vertebral bodies (N.V., positive for alizarin red S staining) normalized for the length of each embryo (L.).
All embryos were examined under a light/fluorescence stereomicroscope (SZX-ZB7 Olympus), acquiring images with a Discovery CH30 camera (Tiesselab), which were analyzed with ISC Capture software to take measurements.

After breeding, zebrafish embryos were washed for five minutes with a methylene blue solution (2 mL of 0.1% methylene blue in 1 L E3 medium) to suppress eventual fungal and mold outbreaks [20 ]. Health conditions of embryos were checked using a light stereomicroscope (Olympus SZX-ZB7) [21 (link)] and grown up to 4 days post-fertilization (dpf) in E3 medium in a dark incubator at 28 °C.

To visualize and quantify the new bone matrix deposited on the scale as new mineralized growing ring, fish have been double stained using two dyes specific for the mineralized matrix, following our previously published protocol [41 (link)]. Briefly, at the beginning of the experiments, fish have been live stained with a 0.005% Alizarin Red S (ARS, Sigma Aldrich) E3 solution overnight in the dark at 28 °C. After 2 weeks, fish have been live stained overnight in the dark with a 0.005% calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, Sigma Aldrich) E3 solution. After repeated washes, scales have been collected as previously described and fixed in 3.5% formaldehyde 0.1 M sodium phosphate buffer solution, then scales have been analysed using a fluorescence microscope (Olympus SZX-ZB7) equipped with a Discovery CH30 camera (TiEsseLab). Scale growing ring and scale area have been measured using ISC Capture Software (TiEsseLab).

Fish were euthanized using a 300 mg/L tricaine methanesulfonate (Sigma Aldrich, St. Louis, MO, USA) solution [25 (link)]. Zebrafish intestines were isolated using sterile scalpels (Paragon^®, Maersk Medical LTD, Sheffield, England) and stored in DNA/RNA Shield (Zymo Research, Irvine, CA, USA). Scales were carefully removed from either side of the fish body using Dumont^® Stainless Steel Forceps (Sigma Aldrich) and processed differently depending on the analysis to be performed as described below. All samples were isolated using a light stereomicroscope (Olympus SZX-ZB7, Olympus, Tokyo, Japan).

Three different experiments were performed on adult fish. First, the 3-month-old young adult zebrafish were treated with 250 nM CHP for 14 days. Double staining with alizarin red/calcein was performed to quantify the growth rate of the mineralized tissue in the scales, as described below. The 9-month-old zebrafish were treated for 14 days with 500 nM CHP with/without 80 μM PN to verify the protective effects of CHP in the glucocorticoid-induced osteoporosis (GIOP) model. Finally, the 9month-old zebrafish were treated with 80 μM PN for 14 days and were then treated for another 14 days with PN with/without 500 nM CHP to verify the CHP curative effects in the GIOP model. All treatment solutions were changed every 48 h. At the end of each treatment, the fish were anaesthetized with 0.16 mg/mL tricaine methanesulfonate [20 ]. The scales were removed using Dumont^® Stainless steel forceps (Sigma Aldrich) from both sides of the fish’s body under a light stereomicroscope (Olympus SZX-ZB7).

Evaluation of the bone matrix profile was performed by direct immersion of the fish in two different staining solutions, the first with alizarin red S, to stain the mineralized matrix present in the scales of the GIOP fish model, and the second with calcein, to highlight the new mineralized matrix deposed during the healing process that closes the resorption lacunae in the scale borders. In detail, fish treated with PN and then used to test HRW healing properties were stained overnight in the dark with 0.005 alizarin red S (Sigma) pH 7.5 E3 solution after 14 days of treatment. After another 14 days with/without HRW, a second staining was performed using 0.005% calcein (Sigma) pH 7.5 solution overnight in the dark. Then, fish were repeatedly washed with E3 medium solution, and scales were explanted, as described before, fixed using 3.4% formaldehyde 0.1M sodium phosphate buffer solution for 15 min and then analyzed using a fluorescence microscope (Olympus SZX-ZB7). Merging the images using ISC Capture Software (TiEsseLab) with red alizarin red S staining and the green signal of calcein staining, it was possible to highlight the new mineralized matrix of the closing resorption lacunae along the scale borders.

To perform the histological ALP assay, scales have been fixed with a 10% formalin 0.05 cacodylate buffer (pH 7.4) solution, rinsed in PBS and then exposed to BCIP®/NBT liquid substrate (Sigma Aldrich) according to manufacturer’s protocol. Histological TRAP activity assay has been performed using Leukocytes Acid Phosphatase (TRAP) Detection Kit according to the manufacturer’s protocol (Sigma Aldrich). Stained scales have been analysed using a stereo-microscope (Olympus SZX-ZB7) equipped with a Discovery CH30 camera (TiEsseLab) to identify TRAP-positive scales and quantify the resorbing area.